RESUMEN
Investigations into the structure-activity relationships (SAR) of a series of phthalazine-based inhibitors of p38 are described. These efforts originated from quinazoline 1 and through rational design led to the development of a series of orally bioavailable, potent, and selective inhibitors. Kinase selectivity was achieved by exploiting a collection of interactions with p38alpha including close contact to Ala157, occupation of the hydrophobic gatekeeper pocket, and a residue flip with Gly110. Substitutions on the phthalazine influenced the pharmacokinetic properties, of which compound 16 displayed the most desirable profile. Oral dosing (0.03 mg/kg) of 16 in rats 1 h prior to LPS challenge gave a >50% decrease in TNFalpha production.
Asunto(s)
Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Ftalazinas/química , Ftalazinas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Animales , Células Cultivadas , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Humanos , Proteína Quinasa 14 Activada por Mitógenos/química , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Modelos Moleculares , Estructura Molecular , Ftalazinas/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Quinolinas/síntesis química , Quinolinas/química , Quinolinas/farmacología , Ratas , Sensibilidad y Especificidad , Relación Estructura-ActividadRESUMEN
Hydrogen bonds involving a carbon donor are very common in protein structures, and energy calculations suggest that Calpha-H...O hydrogen bonds could be about one-half the strength of traditional hydrogen bonds. It has therefore been proposed that these nontraditional hydrogen bonds could be a significant factor in stabilizing proteins, particularly membrane proteins as there is a low dielectric and no competition from water in the bilayer core. Nevertheless, this proposition has never been tested experimentally. Here, we report an experimental test of the significance of Calpha-H...O bonds for protein stability. Thr24 in bacteriorhodopsin, which makes an interhelical Calpha-H...O hydrogen bond to the Calpha of Ala51, was changed to Ala, Val, and Ser, and the thermodynamic stability of the mutants was measured. None of the mutants had significantly reduced stability. In fact, T24A was more stable than the wild-type protein by 0.6 kcal/mol. Crystal structures were determined for each of the mutants, and, while some structural changes were seen for T24S and T24V, T24A showed essentially no apparent structural alteration that could account for the increased stability. Thus, Thr24 appears to destabilize the protein rather than stabilize. Our results suggest that Calpha-H...O bonds are not a major contributor to protein stability.